Ultra high-throughput opti-nanopore DNA readout platform

ABSTRACT

Described herein are methods for analyzing polymer molecules. These methods are employed for the high throughput readout of DNA and RNA molecules with single molecule sensitivity. The method of the present invention comprises (1) the electrically controlled unzipping of DNA (or RNA) double strands, and (2) the readout of the molecule&#39;s identity (or code) using one or more molecule signal detection.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/573,627, accorded a 35 U.S.C. §371(c) date of Aug. 21, 2008, which isa national phase filing under 35 U.S.C. §371 of internationalapplication number PCT/US2005/028566, filed Aug. 12, 2005, which claimsthe benefit of priority to U.S. Provisional Patent Application No.60/601,264, filed Aug. 13, 2004, the disclosures of all of which areincorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 31, 2012, isnamed 42697240.txt and is 5,405 bytes in size.

STATEMENT OF FEDERAL GOVERNMENTAL SUPPORT

This invention was made with government support under grant HG003574awarded by National Institutes of Health. The government may havecertain rights in the invention.

FIELD OF INVENTION

The present invention provides a system for analyzing polymer molecules.In particular, the instant invention is directed to single-strandedpolynucleotide molecule sequencing.

BACKGROUND

Ever since Watson and Crick elucidated the structure of the DNA moleculein 1953, genetic researchers have wanted to find fast and efficient waysof sequencing individual DNA molecules. Sanger/Barrell and Maxam/Gilbertdeveloped two new methods for DNA sequencing between 1975 and 1977,which represented a major breakthrough in sequencing technology. Allmethods in extensive use today are based on the Sanger/Barrell methodand developments in DNA sequencing in the last 23 years have more orless been modifications of this method.

Polynucleotides are polymeric molecules comprising repeating units ofnucleotides bound together in a linear fashion. Examples ofpolynucleotides are deoxyribonucleic acid (DNA) and ribonucleic acid(RNA). DNA polymers are made up of strings of four different nucleotidebases known as adenine (A), guanine (G), cytosine (C), and thymine (T).The particular order, or “sequence” of these bases in a given genedetermines the structure of the protein encoded by the gene.Furthermore, the sequence of bases surrounding the gene typicallycontains information about how often the particular protein should bemade, in which cell types, etc. Knowledge of the DNA sequence in andaround a gene provides valuable information about the structure andfunction of the gene, the protein it encodes, and its relationship toother genes and proteins. RNA is structurally and chemically related toDNA, however, the sugar component of RNA is ribose (as opposed to DNAwhich is deoxyribose) and the base thymine is substituted with uracil.

It is appreciated by those skilled in the art that there is a directrelationship between particular DNA sequences and certain-diseasestates. This fact has encouraged many pharmaceutical companies to investheavily in the field of genomic research in the hope of discovering theunderlying genetic nature of these diseases.

Another reason that sequence information is important is the expectedability to determine an individual's susceptibility to particulardiseases based on his or her genetic sequence. The field of geneticdiagnostics is dedicated to identifying nucleotide sequence elementswhose presence in a genome correlates with development of a particulardisorder or feature. The more information is available about genomicsequence elements observed in the population the more powerful thisfield becomes. Furthermore, the more rapidly information about theprevalence and penetrance of sequence elements in the generalpopulation, as well as the presence of such elements in the genomes ofparticular individuals being tested, the more effective the analysisbecomes.

Yet another reason that sequence information is valuable is that anumber of pharmaceutical companies seek to develop drugs that arecustom-tailored to an individual's genetic profile. The hope is toprovide targeted, potent drugs, possibly with decreased dosage levelsappropriate to the genetic characteristics of the particular individualto whom the drug is being administered.

Most currently available nucleotide sequencing technologies determinethe nucleotide sequence of a given polynucleotide strand by generating acollection of complementary strands of different lengths, so that thecollection includes molecules terminating at each base of the targetsequence and ranging in size from just a few nucleotides to the fulllength of the target molecule. The target molecule's sequence is thendetermined by analyzing the truncated complementary strands anddetermining which terminate with each of four DNA nucleotides. A“ladder” is constructed by arranging the truncated molecules in order bylength, and the terminal residue of each rung is read off to provide thecomplement of the target polynucleotide sequence.

Currently available DNA sequencing systems are very powerful. However,they are limited by their speed, their complexity, and their cost. Thespeed of currently available automated sequencers is limited by theinability of the machines to analyze more than several hundred(typically around 600) nucleotides of sequence at a time. Allowing forthe overlaps needed to piece together correctly strands less than 1000bases longs, the standard sequencing process has to be performed as manyas 70 million times in order to determine the human genome sequence(Technology Review 102(2):64-68 1999 March/April; incorporated herein byreference). At a theoretical rate of even 100 million bases per day itwill take at least a year to sequence the human genome once. With thesetechniques, large-scale sequencing cannot become a clinical tool. Forgenetic diagnostics to become practical in a clinical setting, thesequencing rate will have to be increased by at least three to fiveorders of magnitude.

The complexity of current sequencing technology arises from the need toamplify and modify the genetic molecules being sequenced. Thismodification is carried out either chemically or enzymatically, andamplification is achieved by numerous cycles of heating and cooling. Oneof the more popular ways of amplifying and modifying the DNA to besequenced is using the polymerase chain reaction (PCR). PCR involvessuccessive rounds of denaturing, annealing, and extension using a DNApolymerase and resulting in the exponential amplification of theoriginal strand of DNA. The length of time associated with each part ofthe cycle depends on the fluid volume and the length of DNA to beamplified. Typical times are on the order of 10-30 seconds for thedenaturation step, 5-30 seconds for the annealing step, and 1-4 minutesfor the extension step. This cycle is usually carried out 15 to 30times. Therefore, normal PCR times are one-to three hours depending onthe length of the DNA to be amplified. The fundamental physicalprocesses that constrain the denaturing, annealing, and labeling are thenumber of detectable strands needed, the time needed to carry out thisprocess, and the processivity of the enzyme. This entire process is timeconsuming and requires following involved procedures.

Currently there is a need for a more efficient method for sequencingpolynucleotides. The present invention provides for such a method.

SUMMARY

The present invention is directed towards methods for analyzing polymermolecules. These methods are employed for the high throughput readout ofDNA and RNA molecules with single molecule sensitivity. The method ofthe present invention comprises (1) the electrically controlledunzipping of DNA (or RNA) double strands, (2) the readout of themolecule's identity (or code) using one or more molecule signaldetection, and (3) a controlled slowing down of the nanopore threadingprocess by virtue of the molecular interactions between the nucleic acidand hybridized probes (e.g., strongly interacting probes result infurther decrease in translocation velocity).

The instant invention is directed to a sequencing method that involvesthe conversion of a target polynucleotide, such as DNA, into a designedpolynucleotide polymer (or simply “Design Polymer”). This Design Polymeris encoded by a binary code which is read by a detector, thus providingsequence information reflecting the original target polynucleotide.

In one embodiment of the present invention, a target double-strandedpolynucleotide is converted into a Design Polymer. In one aspect, asingle-strand of the target is processed. The conversion involves thereplacement of each nucleotide base of the target polynucleotide withtwo Design Monomers thereby representing that base as a binary code. Forexample, the base adenine can be represented by 0+0, the base cytosineby 0+1, the base guanine by 1+0, and the base thymine by 1+1. In aparticular aspect, each Design Monomer is a double-strandedpolynucleotide sequence. Each of the Design Monomers represents either a“0” or a “1.” Hence, for each and every base of the targetpolynucleotide, there are two corresponding double-stranded DesignMonomers. In one aspect, the Design Monomers are ligated in such amanner so as to reflect the nucleotide sequence of the targetpolynucleotide, thus forming a Design Polymer. In one aspect, thedouble-stranded Design Polymer is converted to a single-strandedmolecule. The single-stranded Design Polymer is hybridized withappropriate molecular beacons, wherein each molecular beacon comprisesone or more signal molecules and one or more quencher molecules.

In one aspect of the present invention, the Design Polymer comprisesonly two sets of Design Monomers: (1) one set of Design Monomers codefor “0,” and (2) the other set of Design Monomers code for “1.” In aparticular aspect of the invention, only two sets of molecular beaconsare employed. One set of molecular beacons hybridizes to Design Monomersrepresenting “0,” whereas the second set of molecular beacons hybridizesto Design Monomers representing “1.” In one aspect, the two sets ofmolecular beacons comprise unique signal molecules, such that molecularbeacons that hybridize to “0” Design Monomers have a different signalmolecule than the molecular beacons that hybridize to “1” DesignMonomers. The hybridization of molecular beacons to individual DesignMonomers of the Design Polymer forms a Beacon-Design Polymer Complex (orsimply, “BDP complex”).

In one aspect of the instant invention, the molecular beacons comprisesuitable fluorophores as signal molecules. In another aspect, themolecular beacon comprises not only a fluorophore but a quencher aswell. In a particular aspect, a fluorophore is located at the 5′ end ofthe beacon, whereas the quencher is located at its 3′ end.

Methods of the present invention employ electrically driven unzipping ofdouble helical nucleic acids. By using an electrically driven nanoporeunzipping method, control over the unzipping time can be maintained,permitting the use of, for example, self-quenched fluorescence probes,such as the widely used molecular beacons. Advantageously, differentmeasurement schemes can be used in which the fluorescence probes arequenched until the moment of their readout set by the unzipping event.

One embodiment of the invention is directed to a nanopore system. In oneaspect, the nanopore comprises α-Hemolysin. The nanopore of the presentinvention can be used to obtain sequence information of apolynucleotide. The BDP complex can be introduced to the nanopore of thepresent invention. In one aspect, there is a 3′ overhang of the DesignPolymer (of the BDP complex) that is introduced into the nanopore of thepresent invention. This 3′ overhang sequence can penetrate through thenanopore until the double-stranded part of the BDP complex is inapposition with the entry pore. The dimension of the nanopore is suchthat only single-stranded polynucleotides can penetrate, thus thedouble-stranded BDP complex is precluded from entry. A voltage can beapplied across the nanopore to unzip the double-stranded BDP complex.This unzipping of the double strand facilitates the removal of amolecular beacon from the Design Polymer, thus permitting a signalmolecule, e.g., a fluorophore, to elaborate its signal, such asluminescence. Additionally, the unzipping permits entry of thesingle-stranded Design Polymer into and through the nanopore. The signalcan be detected by a suitable detector. After a suitable time, thereleased molecular beacon will self-hybridize thereby quenching its ownsignal.

This a reiterative process, the applied voltage continues to translocatethe unzipped single-stranded Design Polymer through the pore until thenext portion of the single-stranded Design Polymer is completely throughwhereupon a subsequent signal from the next molecular beacon in sequenceis elaborated registering another signal by a suitable detector, and isthen self-quenched when it is released from the BDP complex. In thismanner the entire Designed Polymer can be read.

The present invention also pertains to an optical system that can beused with single-molecule detection. This optical system can be used inconjunction with one or more nanopore systems. The present opticalsystem comprises a custom made flow cell having a nanopore support andtwo electrodes. The electrodes are used to apply an electric fieldrequired for the unzipping process while the support enables thesuspension of a phospholipid bilayer in the proximity of a glasscoverslip thereby enabling imaging of the bilayer using a high-powermicroscope objective. The flow cell is mounted on an XYZ nanopositionerin an inverted microscope and can be mobilized for precision alignmentwith the optical axis using a translation stage.

In the following description of certain embodiments, reference is madeto the accompanying drawings, which form a part hereof, and in which isshown by way of illustration a specific embodiment in which theinvention can be practiced. It is to be understood that otherembodiments can be utilized and structural changes can be made withoutdeparting from the scope of the present invention. In the case of anystructures, it is to be understood that these figures are somewhatsimplified because they do not show all conventional details of thedepicted structures, but only the relevant elements. In addition, whilea particular embodiment is shown here, this is not intended to belimiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the conversion of a targetpolynucleotide to a Design Polymer where (a) is the targetpolynucleotide, (b) is an isolated portion of the target (SEQ ID NOS4-7, respectively, in order of appearance), and (c) is the resultingDesign Polymer.

FIG. 2 is a schematic representation of the cycles involved in theconversion of a target polynucleotide to a Design Polymer where (a) aretwo Design Monomers (SEQ ID NOS 4-6 and 20, respectively, in order ofappearance), (b) is the Design Polymer, and (c) illustrates the cyclesinvolved in converting the target (SEQ ID NOS 8-17, respectively, inorder of appearance).

FIG. 3 is a schematic representation of a molecular beacon where (a) isa linearized representation of the beacon, and (b) represents the beaconself-hybridized;

FIG. 4 is a schematic representation of a Beacon-Design Polymer complex;

FIG. 5 is a schematic representation of the translocation process of theBeacon-Design Polymer with the nanopore where (a) shows the initialsteps involved in this translocation process, and (b) illustrates alater stage of the process;

FIG. 6 is a graph showing the results from unzipping a 10 bp hairpin DNAusing active control, where (a) displays the applied voltage vs time (inmilliseconds), and (b) displays the measured ion current through thepore;

FIG. 7 depicts four distinct steps indicated by (A), (B), (C), and (D)are displayed that occur in a typical analysis of a samplepolynucleotide where the upper panel displays schematic drawings of thetranslocation of a Design Polymer into and through a nanopore system,the middle panel depicts the photon counts/ms vs. time, and the bottompanel depicts the voltage being registered;

FIG. 8 is a schematic drawing of an optical system used in conjunctionwith a nanopore system;

FIG. 9 is an enlarged portion of the optical system depicted in FIG. 8;

FIG. 10 is an SEM image of a ˜30 μm hole fabricated in a thin Teflon®(polytetrafluoroethylene, PTFE) film;

FIG. 11 is a graph showing the first few milliseconds (ms) of a typicalunzipping event performed at a constant force. Where the upper panelshows a controlled voltage applied across the pore while the lower panelshows the pore current resulting from the applied voltage. At t=0 theDNA enters inside the pore;

FIG. 12 is a histogram showing the translocation time distribution of asingle stranded DNA (poly(dA)90 (SEQ ID NO: 18)) measured at V=120 mVand 15° C. The histogram was constructed from ˜1,500 individual events.After tT>tP=0.5 ms (the most probable translocation time), thedistribution is fitted by a single exponential with a time constant of0.32 ms;

FIG. 13 is a graph showing a typical unzipping event for the constantvoltage experiment. The upper panel displays the applied voltage patternand the lower panel is the resulting pore current while the inset showsthe typical distribution of the pore current measured at t=5, ms from˜1000 separate unzipping events;

FIG. 14 is a graph showing the unzipping probability, Punzip as afunction oft, for different unzipping voltage levels for HP1. Thevoltage levels used: (circles) 30 mV, (squares) 60 mV, (triangles) 90mV, (inverted triangles) 120 mV and (diamonds) 150 mV;

FIG. 15 is a graph plotting the dependence of τ_(U) on the unzippingvoltage, V_(U), is plotted in FIG. 5, for HP1 (full circles), HP2(triangles) and HP3 (squares) The characteristic time scales obtainedfrom the exponential fits of PUNZIP (FIG. 4) as a function of VU;

FIG. 16 is a graph showing typical unzipping event under constantloading rate, performed at a constant loading rate of 4.5 V/s where theupper panel shows voltage applied at time t=0, which represents thetriggering of the DNA entry into the pore, while the lower pane showspore current during the controlled voltage ramp; and

FIG. 17 is a graph showing a collection of ˜1500 unzipping events (as inFIG. 6) can be used to obtain the distribution of VU and the mostprobable unzipping voltage VC. Main figure: a semi log plot of VC as afunction of the ramp V&, for HP1 (solid circles) HP2 (triangles) and HP3(squares).

DETAILED DESCRIPTION

The present invention is directed towards methods for analyzing polymermolecules. These methods are employed for the high throughput readout ofDNA and RNA molecules with single molecule sensitivity. The method ofthe present invention comprises (1) the electrically controlledunzipping of DNA (or RNA) double strands, (2) the readout of themolecule's identity (or code) using one or more molecule signaldetection, and (3) a controlled slowing down of the nanopore threadingprocess by virtue of the molecular interactions between the nucleic acidand hybridized probes (e.g., strongly interacting probes result infurther decrease in translocation velocity).

Described herein is the employment of two single-molecule detectionmethods: (1) nanopore detection, and (2) single-molecule signal probing,such as fluorescence probing. Double-stranded polynucleotides, such asdouble helix DNA, can be unzipped using an active control of an appliedelectric field over a nanopore. This unzipping of the double-strandedpolynucleotide produces single-stranded elements that can pass through achannel of the nanopore, while double-stranded portions are precludedfrom entry. The entry of the single-stranded elements of thepolynucleotide thus facilitates its analysis, such as obtaining sequenceinformation.

The methods of the present invention involve the conversion of a targetpolynucleotide molecule into a binary code Design Polymer. A targetpolynucleotide can be obtained from any source. In one aspect, thetarget polynucleotide can be a double-stranded polynucleotide moleculesuch as a double helix DNA. Other polynucleotides are also within thescope of this invention including, but not limited to, RNA and PNAmolecules. The target polynucleotide can be single stranded. The targetmolecule can comprise both natural and modified nucleotide bases. Thetarget can be of any origin. For example, the target can be cDNA or gDNAor man-made (synthesized) sequence used to encode any information in anultra high density format. The target polynucleotide can be partially orcompletely synthesized. The target can comprise nucleotides of naturalorigin including both plant and animal. The target polynucleotide can beof any length. For example, the range in size can be from about 10nucleotide bases to about 100 thousand or greater nucleotide bases.

Referring to FIG. 1, a target polynucleotide 10 is obtained (FIG. 1 a).This target polynucleotide 10 is subjected to biochemical conversion.See, U.S. Pat. No. 6,723,513 to Lexow, the entire teaching of which isincorporated herein by reference. For example, three nucleotides havebeen isolated from the target polynucleotide for illustrative purposes(FIG. 1 b). The three nucleotides are “G,” “T,” and “A” (guanine,thymine, and adenine, respectively). The conversion process will bedescribed using just these three nucleotides.

In this conversion process, each nucleotide is represented by a binarycode using “1” and “0.” So, for example, guanine (G) can be representedby the binary code “1+0”, thymine (T) by “1+1”, adenine (A) by “0+0”,and cytosine (C) by “0+1”. The “1” and “0” of the binary code arerepresented by their own unique polynucleotide referred to as a “DesignMonomer” 12. See, FIG. 1 b. FIG. 1 b provides an example of such DesignMonomers 12. What is depicted in FIG. 1 b is a Design Monomer 12′ for“0” and a Design Monomer 12″ for “1”. (The precise polynucleotidesequence for a Design Monomer can be determined by a practitioner.)Therefore, because the Design Polymer 12 comprises a binary code thereare two Design Monomers 12′, 12″ each representing either “1” or “0” butnot simultaneously both “1” and “0”.

In the example provided in FIG. 1, there is a Design Monomer 12′ for “0”and a Design Monomer 12″ for “1”. Given that each nucleotide isrepresented by a binary code necessitates that each nucleotide isrepresented by two Design Monomers. For example, guanine is representedby the binary code “1+0”, therefore, guanine is represented by theDesign Monomer 12″ for “1” and by the Design Monomer 12′ for “0”.However, the Design Monomers must be in proper sequence in order toreflect the particular nucleotide they are representing. So for guanine,Design Monomer 12″ “1” must be followed by Design Monomer 12′ “0” (usingthe conventional map assignment of 5′→3′).

A Design Polymer 14 comprises Design Monomers 12′, 12″ arrayed in aparticular sequence. See, FIG. 1 c. The Design Polymer's 14 sequencereflects the nucleotide sequence of the original target polynucleotide10 in that the Design Monomers 12′, 12″ are arranged from 5′ to 3′ inthe same order found by their cognate nucleotides in the original targetpolynucleotide 10.

The process presented in FIG. 1 can be repeated for an entirepolynucleotide molecule. The size of a target polynucleotide can bemanipulated for convenience, however, the principles remain the same.

A Design Monomer of the present invention comprises a polynucleotidemolecule. The size of the Design Monomer can vary. In one aspect, thesize of the Design Monomer can range from about ten (10) nucleotides toabout one hundred (100) nucleotides. In another aspect, the DesignMonomer is a double-stranded polynucleotide. The polynucleotide can beDNA, RNA or PNA. The nucleotide constituents of a Design Monomer can benatural, modified, or a combination thereof. They can be selected fromthe group consisting of adenine, cytosine, guanine, thymine, uracil,modifications thereof, and alike. A practitioner can construct DesignMonomers based on the parameters of a given protocol. The key is that atleast two different Design Monomers should be constructed in order toeffectuate the binary code feature of the present invention.

A Design Polymer of the present invention comprises a plurality ofDesign Monomers. The Design Polymer represents a biochemically convertedtarget polynucleotide. The relationship between the Design Polymer andthe original target polynucleotide is such that a practitioner can,using only the Design Polymer, determine the nucleotide sequence of theoriginal target polynucleotide. The Design Monomers of a Design Polymerare linked together, for example, covalently linked, to form apolynucleotide molecule. Again, the sequence of linking the DesignMonomers is such that their linkage sequence reflects the originalsequence of nucleotides in the target.

Hybrid systems are also within the scope of the present invention. Forexample, if the target polynucleotide is an RNA molecule, the DesignMonomers used to construct the binary code can comprisedeoxyribonucleotides. The converse is equally valid. If a practitionerdesires, there can be hybrid constructs. For example, a DesignMonomer(s) can comprise a hybrid of deoxyribonucleotides andribonucleotides. Various hybrid permutations are well within the scopeof this invention.

Referring to FIG. 2, polynucleotides and oligonucleotides (differingonly relative to size) can be processed according to the methoddescribed above. The method described in FIG. 1 can be a reiterativeprocess, thus permitting an oligomer to under conversion into a DesignPolymer. FIG. 2 depicts such a scenario. For this example, an oligomer16 of 21 base pairs is being used to illustrate the method. Oligomers ofvarious sizes are within the scope of the present invention. Forexample, oligonucleotides ranging from about 10 nucleotides to about5000 nucleotides can be processed. As previously stated, polynucleotidesof considerable length can be digested forming shorter oligonucleotidesusing conventional methods well known to those skilled in the art.

FIG. 2 a depicts two Design Monomers, one 12′ represents “0” and theother 12″ represents “1”. Upon closer examination it can be observedthat the two Design Monomers 12′, 12″ comprise different nucleotidesequences. In this particular example, each Design Monomer comprises tenbase pairs. These two Design Monomers 12′, 12″ can now be used torepresent the target nucleotide 16 sequence via a binary code. FIG. 2 billustrates the binary code assignment, also, FIG. 2 b depicts a DesignPolymer 14 formed by linking Design Monomers 12′, 12″ together. Thelength and sequence of the Design Monomers 12′, 12″ is flexible whichpermits the resulting Design Polymer 14 to be tailored made.

FIG. 2 c illustrates the conversion cycling of target to DesignPolymers. The target polynucleotide 16 can be cycled through threeenzymatic steps: A, B, and C. In this illustration, three target basesare converted per cycle. The target polynucleotide 16 in thisillustration comprises 21 base pairs, therefore, a total of seven cyclesis required for full conversion the target. In FIG. 2 c, the upperstrand (bold) is converted in this illustration.

Enzymatic step A comprises the digestion of the target 16 with at leasttwo IIs restriction enzymes, such as NlaIII, in order to generateoverhangs for conversion and Design Polymer linkage. It is wellappreciated by those skilled in the art that digestion of a targetpolynucleotide can be accomplished employing other restriction enzymesknown in the art.

Step B is a litigation step where binary Design Polymer sequences arespecifically attached to the 3′ ends of target fragments having thecorresponding sequence in the 5′ end. This step involves the division oftarget fragments into separate wells where the number of wellscorresponds to the number of permutations of bases being converted(e.g., conversion of three bases requires 64 wells). In each well thereis a Design Polymer sequence and a specific adapter (not shown)corresponding to the sequence that should be converted in thatparticular well (i.e., there will be a non-specific association of theDesign Polymer to the 3′ end of each fragment and a specific ligation ofthe adapter to the 5′ end of the correct fragments). Following ligation,the wells can be pooled into one containing vessel.

Step C is an amplification step. A polymerase chain reaction (PCR) stepis performed to amplify and select those fragments that havesuccessfully been attached to a Design Polymer and a specific adapter.

This conversion method can be performed with massive parallelism wherebillions of different target molecules are converted in the samereaction. See, U.S. Pat. No. 6,723,513.

Once the Design Polymer is formed, one or more signal molecules areassociated with the Design Polymer. In one aspect, the signal moleculeis a molecular beacon. A single-stranded Design Polymer is obtainedthrough means well known to those skilled in the art. Thesingle-stranded Design Polymer is admixed with molecular beacons underconditions suitable for hybridization (e.g., by slow temperaturequenching in the following buffer 100 mM KCl, 1 mM MgCl₂ 10 mM Tris-Hcl,pH 8.0) forming a Beacon-Design Polymer complex (BDP complex).

FIG. 3 depicts schematically a typical molecular beacon 18. (See, TyagiS, Kramer FR. 1996 Molecular beacons: Probes that fluoresce uponhybridization, Nature Biotech. 14:303-8; Bonnet G, Tyagi S, Libchaber A,and Kramer FR. 1999. Thermodynamic basis of the enhanced specificity ofstructured DNA probes. Proc. Natl. Acad. Sci. 96:6171-76). A molecularbeacon (or simply “beacon”) is an oligonucleotide that has a fluorophore(“F”) 20 at one position of the oligonucleotide, e.g., the 5′ end and aquencher (“Q”) 22 at another position of the oligonucleotide, e.g., the3′ end. See, FIG. 3 a. As depicted in FIG. 3 a, the fluorophore 20 canemit a fluorescence signal because it is not encumbered by the quencher22. However, the 5′ end and the 3′ end of a beacon 18 can self-hybridizedue to having complementary bases. See, FIG. 3 b. When the 5′ and 3′ endhybridize or are in close approximation the quencher 22 will quench thefluorophore 20, thus preventing the signal to be elaborated.

In the methods of the present invention, at least two differentmolecular beacons are employed. One molecular beacon will hybridize toDesign Monomers representing “0”, while another molecular beacon willhybridize to Design Monomers representing “1”. Molecular beaconshybridizing to Design Monomer “0” will comprise a signal, such as afluorophore, that is different from a molecular beacon that hybridizesto Design Monomer “1”. For example, molecular beacon “0” (i.e., amolecular beacon hybridizing to a Design Monomer “0”) can havefluorophore “F0” and molecular beacon “1” can have fluorophore “F1” bothof which emit a different and discernable signal.

In one aspect, the percent hybridization between a beacon and a DesignMonomer is about 90% or greater. In another aspect, the percenthybridization of a beacon to a Design Monomer is from about 80% to about90%. In yet another aspect, the percent hybridization of a beacon to aDesign Monomer is about 70% to about 80%. In a further aspect, thepercent hybridization of a beacon to a Design Monomer is from about 60%to about 70%.

FIG. 4 depicts the hybridization of a single-stranded Design Polymer 24with molecular beacon “M1” 18″ or “M0” 18′ forming a BDP complex 24. Inthis BDP complex 24, M1 molecular beacons 18″ hybridize to DesignMonomers “1” 12″, whereas M0 molecular beacons 18′ hybridize with DesignMonomers “0” 12′. As depicted in FIG. 4, M1 molecular beacons 18″ havefluorophore “F1”, whereas M0 molecular beacons 18′ have fluorophore“F0”. These two fluorophores emit a different wavelength signal, e.g.,F1 could emit blue while F0 could emit orange.

Because the present methods involve conversion of a targetpolynucleotide into a binary code-based polymer, i.e., a Design Polymer,the present method requires only two fluorophores. One fluorophorerepresents “0” and the other fluorophore represents “1” of the binarycode “0”+“1”. Those skilled in the art will appreciate that other signalmolecules can be employed consistent with what has been describedherein.

The Beacon-Design Polymer complex can now be introduced to a nanoporesystem. The nanopore system of the present invention, for example,protein channels such as the bacterial alpha-Hemolysin (α-HL), can beused to obtain information on the length and sequence of polynucleotidesand to detect the stochastic binding kinetics of different analytes tomodified portions of the pore. See, Kasianowicz, J., et al., 1996, PNASUSA, 93, 13770-3; Akeson, M., et al., 1999, Biophys. J., 77, 3227-33;Meller, A., et al., 2001, PNAS USA, 97, 1079-1084; and Meller, A., etal., 2001, Phys. Rev. Lett., 86, 3435-38, the entire teachings of whichare incorporated herein by reference. In one aspect, the polynucleotideis a single-stranded molecule such as a single-stranded Design Polymer.It is important to note that a plurality of nanopores can be used thuspermitting the analysis of many Design Polymers, and, therefore, manytarget polynucleotides. In one aspect, the number of pores employed in ananopore system ranges from 5 to about 10. It should be appreciated bythose skilled in the art that many more pores can be employed.

A nanopore system can receive Design Polymers and analyze them basedupon a given protocol. The nanopore system can comprise a variety ofdevices (including detectors) well known in the art. The nanopore systemcan be coupled to molecules, such as receptors, etc.

One skilled in the art will appreciate that chemical channels or porescan be formed in a lipid bilayer using chemicals (or peptides) such asNystatin; ionophores such as A23187 (Calcimycin), ETH 5234, ETH 157 (allchemicals available from Fluka, Ronkonkoma, N.Y.; peptide channels suchas Alamethicin, etc, employing methods well known in the art.

Nanopore systems that are within the scope of the present inventioninclude those described in U.S. Pat. No. 5,795,782, U.S. Pat. No.6,015,714, WO 01/81896 A1, and WO 01/81908 A1, the teachings of whichare incorporated herein by reference.

The nanopore system of the present invention can have a pore moleculesuch as the receptor for bacteriophage lambda (LamB) or alpha-hemolysin.The apparatus used for the nanopore system comprises: (a) anion-conducting pore or channel; (b) the reagents necessary for a DesignPolymer to be characterized; and (c) a recording mechanism to detectchanges in conductance of ions across the pore as the Design Polymerenters and proceeds through the pore. A variety of electronic devicesare available which are sensitive enough to perform the measurementsused in the invention, and computer acquisition rates and storagecapabilities are adequate for the rapid pace of sequence dataaccumulation.

Other pore-forming proteins include Gramicidin (e.g., Gramicidin A, B,C, D, or S, from Bacillus brevis; available from Fluka, Ronkonkoma,N.Y.); Valinomycin (from Streptomyces fulvissimus; available fromFluka), OmpF, OmpC, or PhoE from Escherichia coli, Shigella, and otherEnterobacteriaceae), Tsx, the F-pilus, and mitochondrial porin (VDAC).

Channels and pores useful in the invention can vary (e.g., minimum poresize around 2-9 nm). Pore sizes through which a polymer is drawn will bee.g., approximately 0.5-2.0 nm for single stranded DNA.

There can be multiple pores embedded in the same membrane. Thecombination of many pores (e.g., nanopores) embedded in the samemembrane, or “nanopore array,” the optical readout platform described inthis invention lends itself for considerable parallelism in signalreadout. Investigators have recently shown that using a state of the artElectron Multiplying CCD camera, they can achieve a readout of 256×256pixels area at a frame rate of >200 frames per second. Importantly, ithas been shown that one can resolve light emitted from individualfluorophores in this frame rate. This demonstrates that one can achievea highly paralleled readout. In principle, an array of 100×100 poresfabricated in solid-state film can be imaged using a single CCD camera.Thus, a readout throughput can be boosted by ˜4 orders of magnitudes ascompared to a single nanopore throughput.

The human genome contains ˜3×10⁹ basepairs. A method that enables rapidand inexpensive genome sequencing must be highly paralleled. In oneaspect, the readout speed of the present invention for 500 bases persecond per nanopore will yield total readout of 5×10⁶ nucleotides persecond in a single chip containing 100×100 pores. This amounts to asingle readout of the entire humane genome in ˜10 minutes, an increaseof 4-5 orders of magnitude over state of the art methods.

A modified voltage-gated channel can also be used in the invention(whether naturally or following modification to remove inactivation) andhas physical parameters suitable for e.g., polymerase attachment(recombinant fusion proteins) or has a pore diameter suitable forpolymer passage. Methods to alter inactivation characteristics ofvoltage gated channels are well known in the art (see, e.g., Patton, etal., Proc. Natl. Acad. Sci. USA, 89:10905-09 (1992); West, et al., Proc.Natl. Acad. Sci. USA, 89:10910-14 (1992); Auld, et al., Proc. Natl.Acad. Sci. USA, 87:323-27 (1990); Lopez, et al., Neuron, 7:327-36(1991); Hoshi, et al., Neuron, 7:547-56 (1991); Hoshi, et al., Science,250:533-38 (1990), the entire teachings of which are incorporated hereinby reference).

In one aspect of the present method, the nanopore is α-HL. See, U.S.Pat. No. 6,528,258, the entire teaching of which is incorporated hereinby reference.

The α-HL pore consists of two main parts: a ˜2.5 nm diameter cavity(“vestibule”) and a ˜1.5 nm diameter channel (“stem”), which permeatesthe cell membrane. Double stranded polynucleotide domains can be lodgedin the 2.5 nm vestibule part but they cannot enter the 1.5 nm channel(the diameter of dsDNA is roughly 2.2 nm). Recently it has beendemonstrated that the closing-opening kinetics of short blunt-ended DNAhairpins (from 3 to 8 base pairs) can be directly measured using thenanopore, by lodging the hairpins in the α-HL vestibule, and detectingthe time required for their thermally-activated opening. Upon thespontaneous opening of the hairpin, the single strand DNA enters the 1.5nm channel, causing a brief (but discernable) blockade in the ioncurrent flowing through the pore, thus permitting the detection of thetime interval from hairpin (or double-stranded polynucleotide) lodgingto its first opening.

Referring to FIG. 5, a Beacon-Design Polymer complex 24′ is introducedto the nanopore system 26. As depicted, a single strand 28 of the DesignPolymer 16″ is threaded through a pore 30 of the nanopore system 26. Inone aspect, the single strand portion is the 3′ end of the DesignPolymer 16″. The driving force for movement of the single strand 28Design Polymer 16″ is provided by an electric field that is establishedacross the nanopore 26. Through the use of two electrodes, an electricfield is applied and is used to “unzip” the double-strandedBeacon-Design Polymer complex 24′. This field also provides the drivingforce for entry of a resulting single-stranded Design Polymer 16″ intothe nanopore. As the unzipping occurs, the molecular beacon moieties 32,34 are relieved from their interaction with the Design Polymer 16″ at ornear a channel 30 of the system 26 itself. As the first leading beaconis removed from the BDP complex 24′, the subsequent fluorophore becomesliberated from the quencher of the first beacon, thus the fluorophorecan light up and be detected by an appropriate detector. For example,molecular beacon 32 in FIG. 5 has a quencher 22″, which quenches thesignal of fluorophore F2 20″ that is located on molecular beacon 34.When the first molecular beacon 32 is unzipped and removed from the B-Dcomplex, fluorophore F2 20″ will be freed from the influence of quencher22″, thus it will elaborate its signal. See, FIG. 5 b. The firstmolecular beacon 32 will self-hybridize and consequently its quencher22″ moiety will quench its fluorophore F1 20′. This self-quenchingmechanism minimizes the background noise. This process can be repeateduntil the entire Design Polymer 16″ has been analyzed.

In summary, the foremost fluorophore of the hybridized (to the DesignPolymer) molecular beacon is detected and registered at the same timewhen a voltage ramp is applied in order to unzip (or melt off) thehybridized beacon from the Design Polymer. As discussed above, unzippingoccurs roughly 10 ms after the beginning of the voltage ramp, providingsufficient time for the registration of the fluorophore. The unzippingresults in the release of the first beacon and the immediate exposure(un-quenching) of the next fluorophore hybridized to the Design Polymer.The released beacon will immediately self-hybridize, self quenching itsown fluorophore. The change in the fluorescence intensities (of eitherof the two colors) is detected and triggers the application of the nextvoltage ramp. The cycle repeats until the entire Design Polymer is read.In one aspect, the Design Polymer has a hairpin. The final hairpin isdisposed at the 5′ end of the Design Polymer to force the molecule toenter the nanopore system in one direction only, i.e., thesingle-stranded 3′ end. Upon completion of the translocation of theDesign Polymer into and through the nanopore system, an optical system(discussed below) will sense that the polymer readout has finished (thatthe DNA cleared the pore) when the ion current will rise to the openpore level.

Force-induced unzipping of longer double-stranded helical domains (50bp) of DNA has been demonstrated using the α-HL pore. See, Sauer-Budge,A. F., et al., 2003, Phys. Rev. Lett., 90, 238101, the entire teachingof which is incorporated herein by reference. It was found that thecharacteristic passage time of the molecules, which includes the slidingof the single strand through the pore, plus their unzipping time,follows an exponential dependence on the applied voltage (in the range140-180 mV). Thus the voltage applied to unzip the beacons in thecurrent invention can be used as a sensitive control parameter todetermine the time delay between each unzipping step (of successivebeacons along the DNA). This was shown in detail in our recent data(FIGS. 10-15).

Voltage controlled unzipping is described in Biophysical Journal: MathéJ. et al. 2004 Biophys. J. 87, 3205-12, the entire teaching of which isincorporated herein by reference. This paper demonstrates that shortoligonucleotides (˜10 bases) hybridized to a long complementary singlestranded DNA can be unzipped inside a nanopore in a voltage-controlledmanner, and that the characteristic time scale of the unzipping processstrongly depends on the voltage, further, it can be selected to be inthe range of 1-10 ms. This time scale is highly important, itdemonstrates that: (a) the translocation speed of DNA in a pore can beregulated and, in particular, it can be slowed down by the unzipping ofDNA probes. The amount by which the translocation is slowed down dependsexponentially on the interaction strength between the DNA and thehybridized probed, as well as on the applied voltage. Thus, by selectingthe sequence of probe, different levels of “slowing down” can beachieved. It was also shown that the applied voltage can be used toadjust the unzipping and, thus, translocation speed. Slowing down of theDNA translocation has been a long-standing problem in this field; and(b) investigators were able to slow down the translocation to speedsrange that is compatible with single fluorophore optical detection(0.1-50 ms per base). Without this slowing down, photon noise willdominate the readout wherein a single fluorophore readout cannot berealized using existing technology.

Optical agents for use in accordance with the present inventioncomprise, for example, fluorescent molecules that are quenched whenbrought into close proximity with a quencher molecule. Extensiveliterature is available describing fluorescence and the other types ofcompounds that exhibit this behavior, as well as the particularproperties (e.g., fluorescence decay time, absorbance spectra, emissionspectra, photostability, and quantum efficiency) of these compounds(see, for example, Gilbert et al., Essentials of MolecularPhotochemistry, CRC Press, 1991; Laxowicz, J R, Principles ofFluorescence Spectroscopy (2^(nd) ed.), Kluwer academic 1999; the entireteaching of which is incorporated herein by reference). Detaileddescriptions of fluorescence quenching are available, for example,Kavarnos et al., Chem. Rev. 86:401, 1986; Wagoner, Methods Enzymol.246:362, 1995; Millar, Curr. Opin. Struct. Biol. 6:637, 1996; Petit etal., Biol. Cell 78:1, 1993; the entire teachings of which areincorporated herein by reference.

In recent years, there has been much progress made in the detection ofsingle fluorescent molecules (see, e.g., Mathis et al., Bioimaging5:116-128, 1997; Ha et al., Proc. Natl. Acad. Sci. USA 93:6264-6268,June 1996; Goodwin et al., Acc. Chem. Res. 29:607, 1996; Muller et al.,Chem. Phys. Lett. 262:716, 1996; Sauer et al., Chem. Phys. Lett.254:223, 1996; the entire teachings of which is incorporated herein byreference). Such molecules possess characteristic fluorescence decaytimes that are sensitive to the molecule's electronic environment andtherefore can be quenched by association with polymers that alter theenvironment.

Examples of fluorophores that can be used with the present inventioninclude TMR and Cy5. Other types of fluorescent molecules that can beused are CPM, the Alexa series of fluorescence markers from MolecularProbes, Inc., the Rhodamine family, and Texas Red. Other signalmolecules known to those skilled in the art are within the scope of thisinvention. Numerous other fluorophores can be used in the presentinvention including those listed in U.S. Pat. No. 6,528,258, the entireteaching of which is incorporated herein by reference.

Quenching molecules that can be used in the present invention includeDabcyl, Dabsyl, methyl red and Elle Quencher™. Other quencher moleculesknown to those skilled in the art are within the scope of thisinvention.

The detection system employed in the present invention depends upon theoptical agents being used. The detection system must be capable ofdetecting changes in optical properties of an optical agent on the timescale relevant to the sequencing method described herein. In one aspect,fluorophores are employed as optical agents. A suitable fluorescencedetector therefore would be appropriate. Those skilled in the art arefamiliar with fluorescence detectors.

Referring to FIG. 6, panel (a) displays the applied voltage versus time(in milliseconds). The voltage ramp of the nanopore system is triggeredin real-time analysis of the ion current flowing through a singlenanopore channel as the single-stranded polynucleotide enters the pore.In approximately 0.1 ms after the entry of the single-strandedpolynucleotide into the nanopore (as signaled by an abrupt drop in porecurrent), the voltage is reduced to a “holding” level and then itincreases at a constant rate. FIG. 6 b displays the measured ion currentthrough the pore. After the entry of the single-stranded part of themolecule into the channel, the pore current is reduced to its blockedlevel (˜10% of the open pore current or ˜10 pA). When the voltage rampbegins, the current initially stays at its blocked level, but at t˜10 msthe current abruptly rises to the open pore level because thedouble-stranded polynucleotide unzips and rapidly clears the pore(clearing of the pore lasts less than 0.05 ms). The unzipping voltage(dashed line) can be readily measured.

Referring to FIG. 7, four distinct steps indicated by (A), (B), (C), and(D) are displayed that occur in a typical analysis of a samplepolynucleotide. These steps can be repeated until the sequencing iscompleted. The top panel depicts the entry and transition of a BDPcomplex 24″ through a nanopore channel 30. The middle panel depicts thephoton counts/ms vs. time, and the bottom panel depicts the voltagebeing registered.

At step (A), the readout begins when the single-stranded overhang of theDesign Polymer 28′ enters pore 30′ as it is pulled in by an electricfield. The abrupt reduction in the ion current flowing through the pore30′ due to the entry of the Design Polymer 28′ triggers the readoutby: 1) reducing the voltage from the “driving” level down to the “hold”level and the initiation of the first voltage ramp; and 2) switching on,for example, a laser for fluorescence analysis. Because, the firstbeacon hybridized to the Design Polymer is not quenched, an immediateincrease of its corresponding fluorescence level will be produced,(shown as a light trace in the middle panel). Note that the typicalunzipping time is ˜10 ms. In that time photons emitted from the firstunquenched fluorophore will be registered by an appropriate detector.

Step (B) is initiated immediately after the first beacon 32′ isunzipped. The unzipping of the molecular beacon 32′ will permit thesingle-stranded Design Polymer 16′ to move further inside the nanopore26. This unzipping process results in the un-quenching of subsequentfluorophores by releasing molecular beacons. The released beacons willautomatically self-hybridized due to thermodynamic forces, thus, abeacon's own fluorophore will be quenched. The self-hybridized beaconswill eventually diffuse away. Advantageously, this feature will help toavoid or minimize background noise. The resulting fluorescence signal(middle panel) shows an increase in the red (dark) emissioncorresponding to fluorophore 34, and a decrease in the orange (light)emission corresponding to the loss of and self-hybridization ofmolecular beacon 32′. There is a small (about 50 μs) delay in thequenching of the first beacon 32′ due to the finite time that it takesfor the beacon to self quench. During this small delay time there arefluorescence intensities from both fluorophores 32′ and 34 which can beused to pinpoint the transitions, used to trigger the voltage ramps.

In step (C), there is a similar readout to the first beacon 32′, shownby the lighter line. Note that between steps (C) and (D), where bothbeacons from the same type are read, there is a spike in the orangeintensity (light gray). This is due to the contribution of twofluorophores as explained above. In this way the system reads the entireDesign Polymer. To prevent the entry of the Design Polymer 16′″ into thepore from its 5′ (rather the 3′), a 7 bases hairpin 36 can be addedduring the Design Polymer's 16′″ conversion process. This hairpin 26 iseventually unzipped allowing for complete translocation of the DesignPolymer 16′″ into the inside or vestibule of the nanopore system 26.

The present invention also pertains to an apparatus for determining thesequence of a polynucleotide. An optical system 300 for the detection offluorescence signals from individual DNA molecules, threaded inside amembrane embedded nanopore is described herein.

FIG. 8 is a schematic of optical system 300 that can be used inconjunction with the sequencing method of the present invention wherethe system 300 comprises elements required for detection of opticalsignals. Specifically, FIG. 8 illustrates an optical setup for theoptical detection of single-molecules using one or more bilayermembranes.

The present optical system 300 comprises a custom made flow cell 302comprising a nanopore support 304 and two electrodes 306 and 308.Electrodes 306 and 308 are used to apply an electric field required forthe unzipping process while support 304 enables the suspension of aphospholipid bilayer 310 in the proximity of a glass coverslip 312thereby enabling imaging of the bilayer 310 using a high-powermicroscope objective 314. Flow cell 302 is mounted in an XYZnanopositioner 316 in a custom made inverted microscope and can bemobilized for precision alignment with the optical axis 208 using atranslation stage.

Fluorophores can be excited by an emitted light from a diode laser 318into the optical system 300. The laser 318 can be a 532 nm solid-statelaser (e.g., New Focus™ 3951-20 or Point Source iFLEX 2000™) and coupledwith a single-mode, polarization preserving, optical fiber 320. Thelaser beam is steered via a mirror 321 expanded using an home madeadjustable beam expander (5×) 322, and directed to a back aperture ofobjective 314 using mirrors 324 and 326 and a dichroic mirror 328(Chroma z532 rdc).

The laser beam is slightly expanded at the focal point of objective 314,to allow a larger illumination area. The expansion of the beam isaccomplished by slightly diverging the incoming expanded laser beam atthe entrance to microscope objective 314. By moving one of the lenses onthe beam expander 322 with respect to the other one, the beam isslightly defocused at the focal point of objective 314 and achieves alarger illumination area (˜10 μm). The fluorescence emission iscollected using the same objective 314 and filtered using a long passfilter 330 (Chroma hq560 lp). Light is then imaged either by a frametransfer back illuminated cooled CCD camera 332 or by first focusing thelight onto a pinhole 334 and imaging the pinhole onto two avalanchephotodiode point detectors 336 and 338 (Perkin Elmer SPCM AQR-14) forfast detection. The optical system 300 (excluding the excitation andemission paths) is wholly enclosed in a shielded copper box (not shown)to reduce electromagnetic noise pick up. By using CCD camera 332,several pores will be allowed to be imaged simultaneously, thusmultiplying the throughput of detection system 300.

Instrument control and data acquisition software is used to controlautomatic positioning and imaging of single molecules and for thereal-time nanopore force spectroscopy. The master trigger for both thefluorescence and current data acquisition is the blocked current signalof a single molecule entering nanopore 302. The hardware electronicscomprise three PC boards: a fast digital I/O to interface with the PZTcontroller (Physik Instrumente PI-E710), a photon counter board(National Instruments PCI-6602), and a fast A/D board that samples theion current and produces a programmable voltage gradient across the pore(e.g., National Instruments PCI-6052E).

The instrument is comprised of custom made microfluidic cells used tosupport an approximately 20 μm phospholipid bilayer horizontally, and toexchange buffer solutions. Referring to FIG. 9, a micro-fluidic cell 402is depicted comprising (a) quartz substrate 404, (b) custom machinedPolytetrafluoroethylene (PTFE) film 406, (c) chamber made ofpoly-dimethylsiloxane (PDMS) 408, (d) housing 410, and (e) electrodes412 and 414. A 20-30 μm aperture 416 is fabricated in PTFE film 406 bymechanical imprint. Inset 416 illustrates membrane 418 and the α-HL pore420 containing an RNA molecule 422 where membrane 418 is supported byaperture 416.

FIG. 10 shows an SEM image of ˜30 μm aperture 416 fabricated in PTFEfilm 406. Membrane 418 is suspended in circular aperture 416 within 12μm thick PTFE film 406, approximately 100 μm above the glass coverslip424. This enables high-resolution fluorescence imaging of membrane 418,using a 60×/1.2 N.A. water immersion microscope objective 426 (OlympusUPLAPO60XW). This is in contrast to prior art designs where oilimmersion is used. The use of water immersion objective 426 (high NA)rather than an oil immersion objective, provides a longer workingdistance (˜250 μm). PTFE film 406 separates two small chambers 428 and430 (about 50 μl each) that can be accessed for fluid exchange byspecial tubing. Two Ag/Ag—Cl electrodes 412 and 414 are in contact withthe fluid chambers 428 and 430 and are connected to a high resolutionpatch clamp head-stage amplifier (not shown) (e.g. Axon Instruments 200U) used to apply voltage across the membrane.

Returning to FIG. 9, a nanopore, for example, a α-HL pore 420 isembedded in membrane 418 using a method described in the literature(see, e.g., Meller, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97,1079-1084; Meller, A., et al. (2001) Phys. Rev. Lett. 86, 3435-3438, allincorporated by reference), and the ionic current flowing through pore420 is measured using the patch-clamp amplifier and analyzed inreal-time. As discussed above, cell 402 is mounted on an XYZnanopositioner (e.g., Polytec PI-P527.3CL) to allow precise alignment ofcell 402 and nanopore 420 at the field of view of the objective, and totrack fluorescently labeled nanopore 420 laterally based on real-timeanalysis of the signal.

There are at least two different embodiments of the membrane cell thatare within the scope of this invention. A membrane cell can be made byembedding PTFE film in a custom made PDMS gasket that has an upper fluidchamber and lower fluid chamber. PDMS gasket is housed in a supportingplastic cap. The cell is about 25 mm wide and about 3 mm thick. Itincludes a lower chamber (˜10 μl) that can be accessed using tubing. TheAg/AgCl electrode of the lower chamber and PTFE film are embedded in thePDMS gasket. The cell is covered from the bottom face with glasscoverslip through which imaging is made.

Alternatively, the membrane cell can be composed of two concentriccylinders, an upper chamber and a lower chamber that can slide withrespect to each other by fine micrometers. At the bottom face of upperchamber, PTFE film is fused by heat. The upper chamber is free to slidedown inside lower chamber until the PTFE film approaches the coverslipand can be imaged from below with the microscope. Approximately 20 μmcircular aperture can be fabricated in using a PTFE film which is usedto support a lipid bilayer. The lower chamber is a ˜20 mm wide chamber(6 mm high) and supports a thin glass cover slip disposed at its bottomface. The cell is connected to fluid tubing and two electrodes.

One embodiment of the present invention is directed to quantifying thenumber or amount of a polymer in a given sample matrix. For example, thepolymer can be a polynucleotide. In one aspect, a pre-determined polymeris putatively in the sample. In this aspect at least some portion of thesequence is known. This portion can range from about five to about fiftyor greater nucleotides. Using methods of the present invention, DesignMonomers can be designed to hybridize to the polynucleotide of interestand employing the nanopore system described herein, the targetpolynucleotide can be detected and measured.

Another embodiment of the present invention is directed toward thedetection of a polynucleotide in a sample matrix. Design Monomersspecific for a pre-determined nucleotide sequence can be constructed.These Design Monomers can be introduced to the sample containingpolynucleotides. The sample can be a heterogenous or homogenous sample.The Design Monomers are introduced to the sample under conditionssuitable for hybridization which are well known to those skilled in theart. Using the methods of the present invention, those polynucleotidesthat hybridized to the pre-determined design Monomers can then beisolated and/or processed.

The detection of specific polynucleotide sequences can be effectuated bythe methods of the present invention. For example, the fidelity of thepolymerase used in PCR is not 100% and therefore polynucleotidesproduced using PCR have to be monitored for their fidelity of sequence.Using the methods described herein, a practitioner can verify thefidelity of the polymerase and the overall PCR process. Pre-determinedDesign Monomers can be constructed, reflecting a portion of a targetpolynucleotide, forming a Design Polymer that can be subsequentlysequenced using the present methods.

One embodiment is directed toward the detection of a single or aplurality of mutations in a given polynucleotide sequence. Assuming thatthe target sequence is known in part or in its entirety, Design Monomerscan be constructed such that the examination of a polynucleotidesequence can be accomplished. The Design Monomers can be introduced to asample matrix having a putative target polynucleotide under conditionssuitable for hybridization forming a Design Polymer. This construct canthen be subjected to the methods of the present invention forsequencing. A mutation will be reflected by one or more Design Monomermismatches.

One embodiment of the present invention is directed towards informationstorage. Given that the present invention utilizes a binary code for anypolynucleotide sequence, a nucleotide sequence can be stored using onlythe binary code. In one aspect, a Design Polymer can be designed to codea sequence of numbers (“0” and “1”) just like a computer file. In asense, DNA can be considered a static, ultra dense, storage media. Thenanopore system of the present invention is an efficient means toretrieve (read) the information coded in the sequence.

It should be understood by one skilled in the art that the methodsdescribed herein have numerous practical applications that do not departfrom the scope of the present invention.

EXAMPLE Nanopore Unzipping of Individual DNA Hairpin Molecules Usingα-HL

In order to enable nanopore measurements with time-dependent electricfields such as with a sequencing method, an unzipping method has beendeveloped. The present unzipping method uses high voltage to optimizethe entry rate into a pore, while the unzipping voltage is keptvariable. Thus, hundreds of individual molecules can be probed over ashort period of time (few minutes). The method allows a practitioner torapidly change the electric field applied across the nanopore during thepassage of a polynucleotide.

This unzipping method is particularly advantageous in studying theunzipping kinetics of individual DNA hairpin molecules, over a widerange of voltages from 30 mV to 150 mV and at different loading rates(0.5 V/s-100 V/s). Moreover, the method allows dynamic forcemeasurements in which a constant loading rate is applied rather than aconstant force. These two complementary measurements allow smallenthalpy changes in the DNA hairpin to be resolved down to 1 kcal/mol.

There are three consecutive steps in the nanopore unzipping process ofthe DNA hairpins: (A) the entry and sliding of the single-strand DNAoverhang (50 nt), until the double-stranded (helical) part itself islodged in the 2.5 nm vestibule of the α-HL; (B) the unzipping of thehelical part; and (C) translocation of the unzipped single strand partthrough the pore.

Referring to FIGS. 11-17, the steps were characterized, in order toresolve their typical time scales at different voltages. By using theactive voltage control method, the overhang entry (part A) can bedecoupled from the other steps, and that the translocation time of theunzipped strands (part C) is much shorter than the typical unzippingtime and thus can be neglected.

Firstly, the experiments characterized the time scale associated withsingle-stranded DNA (“ssDNA”) threading in a pore as a function ofvoltage by showing that the typical time scale for unzipping a 10 bphairpin is substantially longer than the threading time of the ssDNA.Next, the unzipping kinetics information for stationary (or stepfunction) voltages in the range 30-150 mV was displayed to determine theeffective charge on the DNA strand inside the pore. Finally, theexperiment demonstrated the dependence of the unzipping kinetics on theloading rate. The data was interpreted using a simple theoretical modelsimilar to the one developed for other single molecule (“SM”) unbindingexperiments.

Materials and Methods

PAGE-purified ssDNA and DNA hairpins (Eurogentec, San Diego, Calif.)were buffered in a 10 mM Tris, 1 mM EDTA, pH 8.5 solution and, prior tothe measurements, were heated to 75° C. for 10 minutes and then quenchedto 4° C. The hairpin molecules consisted of a 3′ single strandedoverhang (50 mer poly-dA (SEQ ID NO: 19)), and a 10 bp helical partcontaining an intervening 6 base loop with the following sequence (theself-complementary parts are underlined):

(SEQ ID NO. 1) HP1: 5′-GCTCTGTTGCTCTCTCGCAACAGAGC(A)₅₀;

In addition, a similar hairpin (HP2) with a single (TT) mismatch on the5th base from the 5′ end, was also prepared with the following sequence:

(SEQ ID NO. 2) HP2: 5′-GCTCTGTTGCTCTCTCGCAACTGAGC(A)₅₀;

and also a 7 bp helix hairpin (HP3), with the following sequence:

HP3: 5′-GTCGAACTTTTGTTCGAC(A)₅₀; (SEQ ID NO. 3)

(The basic apparatus and experimental method used for reconstituting theα-HL channel in a horizontally supported planar lipid bilayer isdescribed above.) The temperature of the system was maintained at15.0±0.1° C., using a custom cell design. The buffer solution was 1MKCl, 10 mM Tris-Hcl, with a pH of 8.5. The α-HL open pore conductanceunder these conditions was 0.82 pS in the forward bias. The ion currentwas measured using a patch-clamp amplifier (Axopatch 200B, AxonInstruments, Union City, Calif.) and the signal was filtered using a 100kHz low-pass 4-poles Butterworth filter (Krohn Hite 3302, Avon, Mass.).The signal was digitized at 1 MHz/22 bits using a DAQ card. All controland acquisition software was written using National Instruments'LabView. The apparatus incorporates a feedback loop used to control theapplied transmembrane voltage. The response time of the membranepotential to a step in the control voltage was 4±1 μs. In eachexperiment (performed at given conditions set by the voltage or thevoltage ramp) data was typically collected over 1,000 unzipping events.The software and hardware combination permits high-throughput unattendeddata acquisition, such that the total acquisition time for eachexperiment was ˜10 minutes.

Each unzipping event consisted of three parts: 1) Threading and slidingthe single stranded overhang until the helical part was lodged insidethe vestibule of the α-HL; 2) Holding the hairpin inside the pore at lowvoltage; and 3) Unzipping the double-stranded DNA.

The first and the second parts were designed to prepare the system forthe third part, such that unzipping always starts at a givenconfiguration of the molecule with respect to the pore.

As with any single molecule experiment, variations between molecules orevents are unavoidable. There were a set of control measurements(described below) and the unzipping experiments were repeated many timesin order to reduce data scatter. The unzipping part consisted of twotypes of measurements: unzipping at stationary force, and unzipping witha fixed loading rate. These experiments are described in the “Results”section.

The voltages and times selected for the first two parts (sliding andholding) were unchanged in all experiments. FIG. 11 shows the first fewmilliseconds (ms) of a typical unzipping event performed at a constantforce. The upper panel displays the applied voltage as a function oftime and the lower panel is the resulting pore current. The event beginswith the threading of the single stranded poly(dA) end inside the pore.The entry of the polynucleotide into the channel is detected by theabrupt decrease in the open pore current, which sets off a trigger inthe acquisition system and defines the t=0 point (dashed line). Themolecule is drawn inside the channel at V=220 mV for a time t_(d)=300μs. This time was chosen to be slightly larger than the most probabletranslocation time, t_(P), of 40 mer DNA, as shown in the results.Therefore, at t=t_(d) most of the DNA hairpins are expected to be fullylodged in the α-HL vestibule. This hypothesis was verified by twoindependent supplementary measurements: in the first experiment t_(d)was varied from 50 to 700 μs and measured the distribution of escapetimes upon the reversal of the voltage to V=−120 mV at t=t_(d). Therewas a monotonic increase in the typical time required for the moleculesto be pulled away from the pore for t_(d) values between 50 and 300 μs.But, for t_(d)>300 μs the curve saturated to a constant level,indicating that the single strand overhang of DNA was threaded throughthe channel and then stopped when the double-stranded helical partentered the vestibule. Further evidence came from the analysis of thelevel of the blocked ion current during t_(d). Specifically, the averagecurrent (for t_(d)=300 μs) displayed two clear peaks: a majorlow-current peak at ˜6 pA and a secondary peak at the normal poly(dA)blocked level (˜11 pA). The lower current peak was attributed toadditional blocking of the pore by the helical part of the hairpinoccupying the α-HL vestibule.

The fraction of unzipped hairpins at t=t_(d)=300 μs was very small. Thisfraction was quantified by measuring the translocation probabilitydistribution of the hairpin molecule at V=120 mV (data not shown). Fromthis distribution it was estimated that the fraction of unzippedhairpins at t=t_(d)=300 μs was smaller than 0.5%. Following the initialsliding of the DNA, at t=t_(d) the voltage was reduced to a low“holding” potential (20 mV) for a time t_(H)=500 μs. This voltage wasfound to be sufficiently large to keep the hairpins in the vestibule,but was too small to induce significant unzipping as evident from thedata displayed below. The choice of 500 μs was dictated by convenience(in a separate set of experiments hairpins were held in the pore for upto 5 seconds). At the end of the holding period the unzipping voltage,V_(U) (or the loading rate)

=dV/dt was applied.

Results

The translocation time distributions of ssDNA.

The translocation time (t_(T)) of each individual DNA is defined as thetime interval from the entry of the first few bases of the DNA moleculeinside the channel part of the α-HL pore, to the exit of the moleculefrom the other side of the channel. These events are clearly observed bythe abrupt reduction of the ionic current flowing through the pore downto ˜10% of the open pore current. In the case of the hairpins, the DNAcan only enter the channel with its single strand 3′ overhang, since theloop at its other end is too large to enter the pore. In this case,t_(T), is a sum of the three consecutive processes described above, i.e.t_(T)=t_(s1)+t_(unzip)+t_(s2), where t_(s1) is the sliding time of thesingle strand overhang (poly(dA)₅₀ (SEQ ID NO: 19)), t_(s2) is thesliding time of the unzipped hairpin+loop (26 bases), and t_(unzip) isthe unzipping time.

FIG. 12 displays the translocation time distribution of a singlestranded DNA (poly(dA)90 (SEQ ID NO: 18)) measured at V=120 mV and 15°C. The histogram was constructed from ˜1,500 individual events. Thedistribution of the ssDNA displays a prominent peak at t_(P)=0.5 ms,which was used to characterize the process. After tT>tP=0.5 ms (the mostprobable translocation time), the distribution is fitted by a singleexponential with a time constant of 0.32 ms. Previous studiesdemonstrated that t_(P) scales linearly with the number of bases (for22-mers and above). Thus, from the translocation distribution of thepoly(dA)₉₀ (SEQ ID NO: 18), it was estimated that t_(s1)˜0.5ms×50/90˜0.28 ms. Notice that this time is much smaller than thecharacteristic t_(T) of the DNA hairpin (˜2-10 ms) measured in a similarway (data not shown). Nevertheless, as is described below, it ispossible to decouple the initial sliding from the unzipping process,thus eliminating t_(s1) completely.

The contribution of t_(s2) to the unzipping process can be estimatedfrom the studies of the translocation time of poly(dA) as a function ofV. Based on these measurements estimate t_(s2)˜660 μs at 30 mV and ˜100μs at 150 mV, for 26 nucleotides. The estimated values of t_(s2) as afunction of the voltage can be subtracted from the total unzipping timemeasured in each event to yield a more accurate estimation of t_(unzip).This correction is small (see below) and did not impact the results.

The measurements of t_(s2) also give an idea of the average sliding timeper nucleotide (e.g., 6 μs at 100 mV). As shown below, this timescale isimportant for the elucidation of the unzipping mechanism inside thepore. When the hairpin is lodged in the pore its unzipping kinetics isaffected by the competition between the re-zipping and sliding processes(an unzipped nucleotide can either rejoin the hairpin or slide along thechannel, thereby blocking re-zipping). If the sliding time is shortcompared to the re-zipping time, re-zipping will be prohibited. Incontrast, if the sliding time is long, the hairpin will undergo manyopening-closing transitions before complete unzipping.

1. Unzipping Kinetics at Constant Force

The unzipping of DNA hairpins under a stationary voltage (or force) wasstudied. An abrupt change in the voltage was applied across the nanoporeand the unzipping kinetics was measured. Typical voltage and currenttraces used in this experiment are shown in FIG. 13. The DNA enters thechannel at time t=0. The molecule is briefly pulled and held in the poreas explained in FIG. 10. At t=0.8 ms, the unzipping voltage is applied(90 mV), but the current is blocked (lower level) until unzipping occursat t=tU=70 ms, as indicated by the abrupt increase in the current. Uponthe application of the unzipping voltage V_(U), the current slightlyincreased to its proper blocked pore level (see FIG. 11 for a zoom inview of the pore current), and stayed at this blocked level until t˜70ms (measured from the application of the unzipping voltage). At thispoint the current abruptly increased to the open pore current levelcorresponding to this voltage. Since the sliding time of the unzippedhairpin is too short to be resolved on the time scale of FIG. 13, thistransition signals the unzipping moment of the hairpin. The unzippingprocess was repeated using an automated procedure which accumulated ˜100separate unzipping events per minute.

The data was analyzed by calculating the probability that an unzippingevent has occurred in the time interval [0−t], where t=0 is defined asthe moment when V_(U) is applied. For this calculation there were ˜1000unzipping events acquired and the average pore current measured in a 50μs time window centered at time t was plotted. The distribution of thecurrents exhibits two well separated peaks associated with the blockedand empty pore states respectively (inset in FIG. 13). By calculatingthe ratio of the number of “empty pore” (high current) events to thetotal number of events, the accumulated unzipping probability at time twas obtained. The voltage levels used: (circles) 30 mV, (squares) 60 mV,(triangles) 90 mV, (inverted triangles) 220 mV and (diamonds) 150 mV.The measurements described above were repeated for different values ofV_(U) (30, 60, 90, 220, and 150 mV) and applied a similar analysisprocedure. The data showing the probability to unzip the 11 hairpin as afunction of the probing time is displayed in FIG. 14. PUNZIP wascalculated as the ratio of the number of events under the high currentpeak to the total number of events (see inset of FIG. 13), for eachprobing time. At short times (i.e. t<1 ms) the unzipping probability isvery small regardless of the amplitude of V. At t˜10 ms there is apronounced difference between the unzipping probabilities at small andlarge V_(U) values. Because the unzipping is immediately followed by thetranslocation of 26 nt (single strand) through the pore, the measuredunzipping time includes two terms: the true unzipping time and thesliding time (t_(s2)) of the 26-mer. The measurements were corrected byestimating t_(s2) as explained earlier. However, because the correctionis much smaller than the unzipping time (˜1%), it had a very smalleffect on the results.

The unzipping probability distributions shown in FIG. 14, were fit bysingle exponential functions, which yielded the characteristic unzippingtime τ_(U), at the different voltage levels. The dependence of τ_(U) onthe unzipping voltage, V_(U), is plotted in FIG. 15, for HP1 (fullcircles), HP2 (triangles) and HP3 (squares), respectively. It is notedthat τ_(U) depends exponentially on V_(U), as apparent from the straightline fits. This dependence is expected from the modified Kramers ratemodel: (24)τ_(U)(V_(U))=τ₀e^((−V) ^(u) ^(/V) ^(β) ⁾, where τ₀=Ae^((E)^(b) ^(/k) ^(B) ^(T)) is the zero voltage transition time, E_(b) is theenergy barrier for dissociation of the hairpin, andV_(β)=k_(B)T/Q_(eff). The slopes of the lines in FIG. 11 give a singlevalue of V_(β)=22±2 mV, which can be used to estimate the effective DNAcharge Q_(eff)≈1.13±0.10e. This charge is associated with roughly 22nucleotides that span the α-HL channel and, therefore, the effectivecharge per nucleotide is: 1.13/12=0.094e. From the intercepts of thefits with the vertical axis, the values of τ_(U)(0) (at zero force) canbe inferred. The experimental results showed τ_(U)(0)=2.1±0.2 s for HP1,1.2±0.1 s for HP2 and 0.34±0.05 s for HP3.

2. Unzipping Kinetics at Constant Loading Rate

With the active control method, an arbitrary time-dependent voltage V(t)can be applied to measure the dynamics of bond breakage. In particular,force spectroscopy measurements are typically performed at a constantloading rate, or ramp

=dV/dt. In the following paragraphs first show the results of aderivation of the expected distribution of the unzipping voltages forany given

, and the dependence of the critical voltage (or the most probableunzipping voltage) on

. The analysis follows the approach of Evans and Ritchie adapted to thenanopore case. The results are consistent with this simplified model forlarge loading rates, but deviate from the model at lower rates. Assumean idealized 1D energy landscape along the direction of the appliedforce, with a single energy barrier. In equilibrium (with no forceapplied), the closed hairpin state is represented as a deep minimum inthe energy landscape, separated from the open state by an energy barrierE_(b). In order to unzip the hairpin, the molecule has to cross thisenergy barrier. In the presence of the biasing voltage (or force) thebarrier is reduced and the Kramers time (τ₀) is modified according toτ(V)=τ₀e^((−V/V) ^(β) ⁾ where τ₀ and V_(β) are as defined earlier. HereV=V(t) is time dependent. The probability per unit time that unzippinghas occurred between t and t+dt is given by

${P(t)} = {{\tau^{- 1}(t)}{{\exp( {- {\int_{0}^{t}\frac{\mathbb{d}t^{\prime}}{\tau( t^{\prime} )}}} )}.}}$This equation can be expressed in terms of V(t)=

t, giving the distribution of the unzipping voltages:

$\begin{matrix}{{p(V)} = {\frac{1}{\tau_{0}V\overset{\;^{\prime}}{\; Y}}\exp\lfloor {\frac{V}{V_{\beta}} - {\frac{V_{\beta}}{\tau_{0}V\overset{\prime}{Y}}( {{\exp( \frac{V}{V_{\beta}} )} - 1} )}} \rfloor}} & (1)\end{matrix}$

The critical unzipping voltage, V_(C), is defined by the maximum of thisdistribution, which is:

$\begin{matrix}{V_{c} = {V_{\beta}\ln\lfloor \frac{\tau_{0}V\overset{\prime}{Y}}{V_{\beta}} \rfloor}} & (2)\end{matrix}$

FIG. 16 displays a typical unzipping event, performed at a constantloading rate of 4.5 V/s. The upper panel depicts the applied voltage andthe lower panel shows the pore current. The unzipping is readilyobservable by the jump of current during the ramp of the voltage, fromthe blocked pore current level to the open pore current level. Theunzipping voltage, VU, is directly obtained from each event. The initialentry of the DNA into the pore was performed as in the previousexperiment (i.e., 0.3 ms sliding and 0.5 ms holding of the moleculeinside the pore). Upon the application of the voltage ramp, the currentremains at the blocked level, but at t˜22 ms (measured from thebeginning of the ramp), the unzipped strand rapidly slides through thepore and there is a sharp increase in the pore current. From the curvethe unzipping voltage, V_(U), (130 mV in this case) can be directlymeasured. The sliding time (t_(s2)) makes the apparent unzipping time(and thus V_(U)) slightly longer. However, as discussed above, this is avery small effect: in the case displayed here t_(s2)˜0.12 ms and thusthe correction is 0.12/22=0.005, a small fraction of the observed time.

In order to obtain sufficient statistics the unzipping experiment wasrepeated at least 1,000 times for any given ramp value. A typicaldistribution of the measured V_(U) values is given in the inset of FIG.17. The distribution is well approximated by Eq. 1 (solid line in theinset). The peak of the distribution is the most probable unzippingforce (or the critical voltage, V_(C)). The measurements were repeatedto obtain the dependence of V_(C) on the voltage ramp in the range0.5-100 V/s, for HP1, for the hairpin with the single mismatch, HP2 andfor HP3 (7 bp helix). The data is displayed on a semi-log plot in FIG.14 (circles, triangles and squares for HP1, HP2 and HP3 respectively).At medium and high voltage ramps (5-100 V/s), V_(C) follows thelogarithmic dependence on

predicted by Eq. 2. According to Eq. 2, the slope of the straight linesin FIG. 17 is simply V_(β). From the logarithmic fit, V_(β.)=24.7±1.0 mVfor HP1, 22.5±1.1 mV for HP2, and 23.3±1.9 mV for HP3, in good agreementwith the constant voltage measurements described above. As expected fromthe fact that V_(β) depends only on the effective charge of DNA insidethe pore, all the molecules yielded almost the same slope. Notice thatat the lower ramp regime, the data deviates from the simple logarithmicdependence predicted by Eq. 2. This deviation is discussed below.Fitting the data (for ramp >1.6 V/s) to equation 2 yields τ_(U)(0)=0.72s and 0.49 s for HP1 and HP2, respectively, values that are smaller thanthose obtained using the constant voltage method.

Previous studies have demonstrated that the closing-opening kinetics ofshort blunt-ended DNA hairpins, lodged in the 2.4 nm α-HL vestibule, canbe described as a single-step process, yielding time scales thatcorrespond to jumps over energy barriers close to the calculated freeenthalpies of the entire hairpin. In these experiments little or noforce at all was applied to denature the molecules since the blunt-endedDNA could not enter the α-HL channel portion. In the currentexperiments, single stranded overhangs extending one end of the DNAhairpins were threaded inside the channel, thus biasing the hairpins'kinetics, presumably by applying force on that strand.

The active control method has allowed the extension of the unzippingmeasurements performed at 220 mV or above to smaller voltages, thusfilling the gap between the zero force and strong force limits. At anygiven voltage V, there can be an estimation of the force applied on thatDNA strand by Q_(eff)V/d where Q_(eff) is the effective charge of thessDNA inside the channel, and d˜5 nm is the channel length. Note thateven at relatively small voltages (small biasing forces), the fastreannealing of thermally “melted” base-pairs can be blocked by thealmost-equally fast progression of the unpaired strand inside the pore.The characteristic ssDNA sliding time was estimated from thetranslocation experiments. This “ratchet” mechanism can split theunzipping of the entire hairpin into several consecutive steps. Thus the15 total unzipping time in this case is expected to be significantlyshorter as compared to the zero bias kinetics.

The experiments show that the characteristic unzipping time measured ata constant force (τ_(U)) decays exponentially with the unzipping voltagelevel, V_(U). The slope of the straight lines fits and was found to beindependent of the hairpin sequence (and thus their enthalpy), andallowed the estimation the effective charge on the ssDNA fragment insidethe channel. The intercepts of the exponential fits to V_(U)=0 provideestimates of τ_(U)(0). It is interesting to compare these values withthe calculated equilibrium time scales associated with fully closed tofully denatured transitions of the hairpins. The mfold server was usedat 1 M Na+, to obtain ΔG⁰=−16.4 kcal/mole for HP1 and −12.2 kcal/molefor HP2, yielding approximated (complete closure to open) time scales ofhours. In contrast, the estimate yielded τ_(U)(0)˜1 s, several orders ofmagnitude shorter. This difference in time scales is in line with the“ratchet” idea presented above. Specifically, if nanopore unzippingtakes place through two thermally activated steps (each of ˜5 bases),rather than one, the total time will be reduced to the order of seconds.

The effective charge on the ssDNA inside the 1.5 nm α-HL channel wasestimated by the two independent approaches, the constant force or thefixed loading rate. Both methods yielded an effective charge of 1.13efor the strand inside the pore or 0.094e per nucleotide, indicating thatthe negative charge of the DNA in the channel is effectivelycounter-balanced by “condensation” of positively charged potassium ionsand by the presence of polar groups on the inner walls of the α-HLchannel, in agreement with previous results. This effective charge canbe used to calculate the force at any given V.

The dynamic force measurements concur with the picture presented above.At high loading rates (ramp >2 V/s) the unzipping time (and thus V_(C))is determined by the rapid change in the potential barrier height due tothe force. In this limit the system does not undergo manyopening-closing transitions and V_(C) is directly proportional to log(

). For small values of

there is a soft crossover to another regime, characterized by weakdependence of the critical voltage on the loading rate. In this regimethe voltage remains sufficiently low for long enough time to allow thesystem to fluctuate between closed and open states, before the eventualunzipping.

The slope of the logarithmic curves in FIG. 17 gives V_(β) as defined inequation 2. The fit parameters are in excellent agreement with the valueobtained using the constant force method. In addition, the extrapolationof the fit to

=1 V/s (i.e., log(

)=0), can be used to estimate τ_(U)(0) for the two hairpins. The valuesobtained from the dynamic measurements are consistently smaller than theextrapolated values obtained at constant force (roughly by a factor of2). Notice that the curves corresponding to HP1 and HP2 are displaced byroughly 20 mV. Using the measured effective charge on the DNA, thischange can be translated to an energy difference of ˜1 k_(B)T (˜0.6kcal/mol).

Although the invention has been described with respect to variousembodiments, it should be realized this invention is also capable of awide variety of further and other embodiments within the spirit andscope of the appended claims.

What is claimed:
 1. A composition comprising: a polynucleotide polymercomprising a plurality of oligonucleotides joined together in a sequencecorresponding to the nucleotide sequence of a target polynucleotide,wherein one or more oligonucleotides in the polynucleotide polymercorrespond to one or more predetermined nucleotide bases of the targetpolynucleotide; a plurality of quenchable fluorescent probes, eachquenchable fluorescent probe comprising a nucleotide sequence, afluorophore and a quencher, wherein: each quenchable fluorescent probeis only hybridized to one of the oligonucleotides in the polynucleotidepolymer; and each of the fluorophores, except a fluorophore of one ofthe terminal quenchable fluorescent probes, is quenched by a quencher ofan adjacent quenchable fluorescent probe.
 2. The composition of claim 1,wherein the target polynucleotide is selected from the group consistingof DNA and RNA.
 3. The composition of claim 2, wherein the targetpolynucleotide is DNA.
 4. The composition of claim 3, wherein the DNA iscDNA or gDNA.
 5. The composition of claim 1, wherein the nucleic acid inthe quenchable fluorescent probe comprises DNA, RNA and/or PNA.
 6. Thecomposition of claim 1, wherein the oligonucleotides comprise DNA, RNAand/or PNA.
 7. The composition of claim 6, wherein the oligonucleotideshave a range in nucleotide bases ranging from about 5 nucleotide basesto about 20 nucleotide bases.
 8. The composition of claim 1, whereineach nucleotide in the target polynucleotide is represented by a binarycode of oligonucleotides.
 9. The composition of claim 8, wherein thenucleotide in the target polynucleotide is selected from the groupconsisting of adenine, cytosine, uracil, guanine, thymine andmodifications thereof.
 10. The composition of claim 1, wherein thecomposition comprises a plurality of different quenchable fluorescentprobes, each probe comprising a different fluorophore, wherein eachprobe is hybridized to a different oligonucleotide.
 11. The compositionof claim 10, wherein the composition comprises two different quenchablefluorescent probes.
 12. The composition of claim 1, wherein thequenchable fluorescent probe self-quenches in isolation.
 13. Thecomposition of claim 12, wherein the quenchable fluorescent probe is amolecular beacon.
 14. The composition of claim 13, wherein a portion ofthe nucleotide sequence on the 5′ end of the molecular beacon iscomplementary to a portion of the nucleotide sequence on the 3′ end ofthe molecular beacon.
 15. The composition of claim 1, further comprisinga nanopore system.
 16. The composition of claim 15, wherein the nanoporesystem comprises a plurality of nanopores and one or more detectionsystems.
 17. The composition of claim 16, wherein a nanopore in thenanopore system is selected from the group consisting of α-hemolysin,receptor for bacteriophage lambda, gramicidin, valinomycin, OmpF, OmpC,PhoE, Tsx, F-pilus, and mitochondrial porin.
 18. The composition ofclaim 17, wherein the nanopore is α-hemolysin.
 19. The composition ofclaim 18, wherein the α-hemolysin comprises a vestibule and a channel.20. The composition of claim 19, wherein the vestibule is about 2.5 nmin diameter.
 21. The composition of claim 20, wherein the channel hasabout a 1.5 nm diameter.
 22. The composition of claim 21, wherein thechannel has a pore size ranging from about 0.5 nm to about 2.0 nm. 23.The composition of claim 15, wherein the nanopore system comprises oneor more nanopores fabricated in a polytetrafluoroethylene thin film. 24.The composition of claim 23, wherein the nanopore size is from about 0.5nm to about 9 nm.
 25. The composition of claim 15, wherein the nanoporesystem comprises a nanopore array.
 26. The composition of claim 15,wherein the nanopore system comprises one or more channels and a lipidbilayer.